Although fungal exposure has often been associated with allergic, toxic health effects and airway inflammation, the actual potential of different fungi or fungal agents to cause or aggravate such adverse effects remains unclear, partly because efforts to establish these causal relationships are frequently weakened by the lack of adequate exposure assessment methods for relevant fungal contaminants. In particular, health effects associated with exposure to secondary fungal metabolites including mycotoxins (particularly those demonstrated to be harmful to humans) have been understudied. Since 2000, NIOSH has conducted a number of epidemiologic studies on respiratory and non-respiratory health in occupants of damp/moldy buildings, and collected more than 2,300 floor/chair dust samples which have been stored for further analysis. The existing database from those studies includes measurements on culturable fungi and bacteria, fungal cell wall components (ergosterol and glucan), bacterial cell wall components (endotoxin and peptidoglycan) as well as occupants? health. Significant associations between various adverse respiratory and non-respiratory health effects and exposure to fungal biomarkers have been demonstrated from NIOSH studies. This project examines associations of exposure to mycotoxins with occupants? health by building the capability of analyzing indoor environmental samples for multiple mycotoxins in a single sample analysis. As part of a collaboration with a research group in Austria, NIOSH completed assays screening more than 550 metabolites in the 500 dust samples collected from a study of 50 elementary schools in a large city. Statistical models will be applied to the data to assess the contributions from fungal metabolites and other potential risk factors or confounders for respiratory and non-respiratory illnesses such as demographics, smoking habit, and exposures to endotoxin, allergens, ergosterol, and fungal and bacterial diversity. Work history data for 1,239 teachers have been cleaned for assigning exposures. Study results will provide an understanding of the effects of exposure to secondary metabolites on respiratory or non-respiratory health for the school staff and may inform potential mechanistic studies. From the study of screening 550+ metabolites, NIOSH identified 174 secondary metabolites that can originate from various sources, with the majority coming from fungi. NIOSH also observed that 17.2% of the samples contained deoxynivalenol that can be released from Fusarium graminearum (sexual stage, Gibberella zeae) and several other Fusarium species. We found Fusarium graminearum from 240 (48%) of the 500 dust samples from DNA ITS sequencing. In addition to deoxynivalenol, mycotoxins such as sterigmatocystin, citrinin, and roquefortine C were found to be present, but in less than 10% of samples. These metabolites can be released from Aspergillus or Penicillium species. With the help of an international collaborator, NIOSH has developed an extraction and analysis method for simultaneously quantifying 36 secondary metabolites in dust samples using UPLC-MSMS. The initial method was further refined with nine C13 isotope-labeled internal standards to compensate matrix effects and has now been applied to analyze 500 dust samples for metabolites from the school study. A previous NIOSH study with spiked samples found that dust sample matrix severely suppressed ionization of the metabolites in the mass spectrometer by more than 80%, resulting in substantial underestimation of the true amount of the metabolites. The 500 sample analysis data have been compiled, cleaned, and processed for data analyses. To compare the results analyzed with isotopically-labeled internal standards which are expensive, NIOSH is in the process of developing a protocol using a standard addition method which is relatively cheap. From the sample storage experiment, NIOSH also found that concentrations of many metabolites and mycotoxins declined more than 50% when stored at room temperature within a year. Efforts to study the stability of standard materials of fungal metabolites and mycotoxins stored (up to one year) in two temperature conditions are ongoing.